Affinity chromatography can be applied to the purification of tagged proteins expressed from any recombinant system including proteins expressed in bacteria, insect and mammalian cells or secreted from yeast. Typically, DNA encoding a peptide sequence such as an antibody epitope or a hexahistidine motif is engineered in-frame with the gene to be expressed and purified. Following expression, the tagged protein is immobilized on a matrix that specifically interacts with the chosen tag allowing the removal of non-specific proteins through a series of washing steps. Elution of the purified protein is often accomplished by the use of competitive peptides or imidazole displacement of the tagged protein from antibody conjugates or nickel resins, respectively.
An advantage that affinity chromatography offers, over traditional chromatography such as anion/cation exchange, size exclusion or hydrophobic chromatography, is that development of purification procedures for individual recombinant proteins is simplified. However, the expense of many affinity matrices, particularly those coupled to antibodies, precludes processing large volumes of cell lysate or spent culture medium cost-effectively.
Carbohydrates represent an abundant and relatively cost-effective chromatography reagent where the effectiveness of a particular material is determined by specific carbohydrate-binding proteins or domains that bind to these reagents. Examples include amylose to which maltose-binding protein binds (U.S. Pat. No. 5,643,758) and chitin to which chitin-binding domain (ChBD) binds (U.S. Pat. Nos. 5,643,758, 6,897,285, 6,984,505, and 7,060,465, and U.S. international application Pub. Nos. WO2006/041849, WO2006/0035333, and WO2005/0227326). The uses of carbohydrates in affinity chromatography depend on the binding affinity between binding proteins and carbohydrates. For some applications, it is desirable that the carbohydrate-binding molecule binds tightly to the carbohydrate where in other applications, it is desirable that the carbohydrate bind reversibly to the carbohydrate-binding molecule so that the carbohydrate-binding molecule may be readily eluted. A method for generating mutant carbohydrate-binding molecules with the desired binding affinity would enable the generation of a range of carbohydrate-binding molecules suitable for different applications.